Biosensors using an enzyme that specifically reacts with a particular substrate are being actively developed in various industrial fields. As for a glucose sensor, which is one of the biosensors, in particular, measurement methods and devices utilizing such methods are being actively developed mainly in medical fields. For example, the glucose sensor has a history of about 40 years since Clark and Lyons first reported about a biosensor including glucose oxidase and an oxygen electrode in combination in 1962 (L. c. Clark, J. and Lyonas, C. “Electrode systems for continuous monitoring in cardiovascular surgery.” Ann. n. y. Acad. Sci., 105: 20-45).
Thus, the adoption of glucose oxidase as an enzyme for the glucose sensor has a long history. This is because glucose oxidase shows high substrate specificity for glucose and superior thermal stability, this enzyme can further be produced in a large scale, and its production cost is lower than those of other enzymes. The high substrate specificity means that this enzyme does not react with a saccharide other than glucose, and this leads to an advantage that accurate measurement can be achieved without error in measurement values. Further, the superior thermal stability means that problems concerning denaturation of the enzyme and inactivation of its enzymatic activity due to heat can be prevented, and this leads to an advantage that accurate measurement can be performed over a long period of time.
However, although glucose oxidase has advantages as described above, it has a problem that the enzyme is affected by dissolved oxygen and this affects measurement results.
Meanwhile, in addition to glucose oxidase, a glucose sensor utilizing glucose dehydrogenase (hereinafter referred to as “glucose dehydrogenase” or “GDH”) has also been developed. This enzyme is also found in microorganisms. For example, there are known glucose dehydrogenase derived from Bacillus (EC 1.1.1.47) and glucose dehydrogenase derived from Cryptococcus (EC 1.1.1.119).
The former glucose dehydrogenase (EC 1.1.1.47) is an enzyme that catalyzes a reaction of β-D-glucose+NAD(P)+→D-δ-gluconolactone+NAD(P)H+H+, and the latter glucose dehydrogenase (EC 1.1.1.119) is an enzyme that catalyzes a reaction of D-glucose+NADP+→D-δ-gluconolactone+NADPH+H+. The aforementioned glucose dehydrogenases derived from microorganisms are already marketed.
These glucose dehydrogenases have an advantage that they are not affected by dissolved oxygen in a measurement sample. This leads to an advantage that accurate measurement can be achieved without causing errors in measurement results even when the measurement is performed in an environment in which the oxygen partial pressure is low, or a high-concentration sample requiring a large amount of oxygen is used for the measurement.
However, although conventional glucose dehydrogenase is not affected by dissolved oxygen, it has problems of poor thermal stability and substrate specificity poorer than that of glucose oxidase. For an enzyme which is used in a sensor, an enzyme that overcomes disadvantages of both of glucose oxidase and glucose dehydrogenase has been desired.
The inventors of the present invention reported results of their studies about GDH using samples collected from soil near hot springs in Sode, K., Tsugawa, W., Yamazaki, T., Watanabe, M., Ogasawara, N., and Tanaka, M., Enzyme Microb. Technol., 19, 82-85 (1996); Yamazaki, T., Tsugawa, W. and Sode, K., Appli. Biochemi. and Biotec., 77-79/0325 (1999); and Yamazaki, T., Tsugawa, W. and Sode, K., Biotec. Lett., 21, 199-202 (1999). The microorganisms in those samples produce a coenzyme-binding GDH, and the enzymologic properties such as optimum reaction temperature, thermal stability, and substrate specificity have already been clear (See the aforementioned documents). This enzyme is a hetero oligomeric enzyme that is constituted by a catalyst subunit having a high thermal resistance (α subunit), an electron transferring subunit (β subunit), and γ subunit having an unknown function, and the activity peaks thereof are observed at 45° C. and 75° C., respectively. Further, the γ and α subunit genes have been cloned, and it has been clarified that the aforementioned microorganism belongs to Burkholderia cepacia, and the N-terminal amino acid sequence of the β subunit has been clarified (Ken Inose, Tokyo Agricultural Engineering University Master's Thesis (2001)). However, the structure of the β subunit gene has not been reported.